Timeline of Achieving a Differentiated RPE Phenotype and Specialised Metabolism of ARPE-19 Cells Under Physiologically Relevant-Inducing Culture Conditions

Background. ARPE-19 cells are widely used to investigate and model retinal pigmented epithelium (RPE). Naïve ARPE-19 cells do not fully replicate RPE features. Previous reports show that under defined conditions, ARPE-19 differentiate towards an RPE phenotype, with protocols relying on prolonged or shorter-term cultures, the latter of which do not achieve full pigmentation. In particular, bioenergetics and mitochondrial dynamics have been assessed in a limited manner to date. The present work aims to characterise ARPE-19 under long-term physiologically relevant phenotype-inducing culture conditions, monitoring the timeline of RPE phenotype acquisition, with a focus on mitochondrial function and bioenergetics alongside structural and functional markers. Methods. Low passage ARPE-19 were cultured for 4 months using low-serum, pyruvate and nicotinamide-supplemented DMEM. Monthly assessments measured pigmentation, TER, gene/protein expression, mitochondrial parameters, and metabolism. Results. ARPE-19 acquired relevant features early (1 month), including polarisation, expression of visual cycle and mitochondrial dynamic drivers, as well as increases in mitochondrial potential, mass/area and branching. Melanin accumulation was increased beginning at 1 month. Bioenergetic assays revealed a shift towards increased reliance on oxidative phosphorylation compared to glycolysis. Conclusions. Under these conditions, ARPE-19 acquired specific RPE features supporting functional assays after 1 month. Time-dependent accumulation of RPE-like features suggests that, for certain applications, later differentiation times are suitable. Appropriately differentiated ARPE-19 are a viable model to study key RPE physiological functions when accompanied by evidence of specific phenotypic and metabolic characterization.